The present invention relates to compounds, their salts, pharmaceutical compositions comprising them, processes for making them and their use in treating Alzheimer""s Disease.
Alzheimer""s Disease (AD) is characterised by the abnormal deposition of amyloid in the brain in the form of extra-cellular plaques and intra-cellular neurofibrillary tangles. The rate of amyloid accumulation is a combination of the rates of formation, aggregation and egress from the brain. It is generally accepted that the main constituent of amyloid plaques is the 4 kD amyloid protein (xcex2A4, also referred to as Axcex2, xcex2-protein and xcex2AP) which is a proteolytic product of a precursor protein of much larger size. The ragged NH2- and COOH-termini of the native Axcex2 amyloid indicates that a complex mechanism of proteolysis is involved in its biogenesis.
The amyloid precursor protein (APP or Axcex2PP) has a receptor-like structure with a large ectodomain, a membrane spanning region and a short cytoplasmic tail. Different isoforms of APP result from the alternative splicing of three exons in a single gene and have 695, 751 and 770 amino acids respectively.
The Axcex2 domain encompasses parts of both extra-cellular and transmembrane domains of APP, thus its release implies the existence of two distinct proteolytic events to generate its NH2- and COOH-termini. At least two secretory mechanisms exist which release APP from the membrane and generate the soluble, COOH-truncated forms of APP (APPs). Proteases which release APP and its fragments from the membrane are termed xe2x80x9csecretasesxe2x80x9d. Most APPs is released by a putative xcex1-secretase which cleaves within the Axcex2 domain (between residues Lys16 and Leu17) to release xcex1-APPs and precludes the release of intact Axcex2. A minor portion of APPs is released by a xcex2-secretase, which cleaves near the NH2-terminus of Axcex2 and produces COOH-terminal fragments (CTFs) which contain the whole Axcex2 domain. Finding these fragments in the extracellular compartment suggests that another proteolytic activity(xcex3-secretase) exists under normal conditions which can generate the COOH-terminus of Axcex2.
It is believed that xcex3-secretase itself depends for its activity on the presence of presenilin-1. In a manner that is not fully understood presenilin-1 appears to undergo autocleavage.
The compounds of the present invention are useful for treating AD by inhibiting the activity of the putative xcex3-secretase thus preventing the formation of insoluble Axcex2 and arresting the production of Axcex2. Further, some of the present compounds also stabilise full-length presenilin-1.
In a further aspect some of the compounds of the present application are useful as inhibitors of presenilin-1 cleavage.
The compounds of the present invention are related to HIV protease inhibitors described in EP-A-337 714 and EP-A-356 223, both in the name of Merck and Co., Inc.. These compounds are aspartyl protease inhibitors. Specifically, a subset of the compounds of the present invention differ from those previously described by the stereochemistry of a hydroxyl group which is a particularly preferred feature of the present invention and has not previously been disclosed for these particular compounds. This is a surprising feature giving rise to the present invention.
The present invention, in one aspect, provides a compound comprising the group: 
wherein R2 and R3 are as defined below, which compound is a diastereoisomer of a known protease inhibitor comprising the group 
wherein R2 and R3 are as defined below.
The present invention accordingly provides a compound of formula I or a pharmaceutically acceptable salt thereof: 
wherein:
R1 is (1) C1-10alkyl, C2-10alkenyl or C2-10alkynyl optionally substituted with one to three substituents independently chosen from:
(i) hydroxy;
(ii) carboxy;
(iii) halogen;
(iv) C1-4alkoxy;
(v) C1-4alkoxycarbonyl;
(vi) xe2x80x94NR6R7 wherein R6 and R7 are independently chosen from hydrogen, C1-5alkyl and C1-5alkoxyC1-5alkyl;
(vii) xe2x80x94CONR6R7 or OCONR6R7 wherein R6 and R7 are independently as defined above;
(viii) xe2x80x94N(R8)QR9 wherein:
Q is C(O), C(S), SO2 or C(NH2);
R8 is hydrogen or C1-4alkyl; and
R9 is hydrogen, C1-4alkyl, C1-4alkoxy, amino, C1-4alkylamino di(C1-4alkyl)amino wherein each alkyl group is independently chosen;
(ix) C3-7cycloalkyl;
(x) phenyl or naphthyl; a five-membered heterocyclic ring containing 1, 2, 3 or 4 heteroatoms independently chosen from O, N and S, at most one of the heteroatoms being O or S; a six-membered heterocyclic ring containing 1, 2 or 3 nitrogen atoms; each of which is optionally substituted by one to three groups independently chosen from:
(a) halogen, cyano and nitro,
(b) hydroxy,
(c) C1-4alkyl, C2-4alkenyl and C2-4alkynyl,
(d) C1-4alkoxy,
(e) NR6R7 wherein R6 and R7 are independently as defined above,
(f) CO2R8 wherein R8 is independently as defined above,
(g) CONR6R7 or OCONR6R7 wherein R6 and R7 are independently as defined above,
(h) SO2NR6R7 wherein R6 and R7 are independently as defined above,
(i) CH2NR6R7 wherein R6 and R7 are independently as defined above,
(j) N(R8)COR8xe2x80x2 wherein R8 is independently as defined above and R8xe2x80x2 is independently as defined for R8, and
(k) NR8SO2R8xe2x80x2 wherein R8 and R8xe2x80x2 are independently as defined above; or
(2) phenyl or naphthyl; a five-membered heterocyclic ring containing 1, 2, 3 or 4 heteroatoms independently chosen from O, N and S, at most one of the heteroatoms being O or S; a six-membered heterocyclic ring containing 1, 2 or 3 nitrogen atoms; each of which is optionally substituted by one to three groups independently chosen from:
(a) halogen, cyano and nitro,
(b) hydroxy,
(c) C1-4alkyl, C2-4alkenyl and C2-4alkynyl,
(d) C1-4alkoxy,
(e) NR6R7 wherein R6 and R7 are independently as defined above,
(f) CO2R8 wherein R8 is independently as defined above,
(g) CONR6R7 or OCONR6R7 wherein R6 and R7 are independently as defined above,
(h) SO2NR6R7 wherein R6 and R7 are independently as defined above,
(i) CH2NR6R7 wherein R6 and R7 are independently as defined above,
(j) N(R8)COR8xe2x80x2 wherein R8 and R8xe2x80x2 are independently as defined above, and
(k) NR8SO2R8xe2x80x2 wherein R8 and R8xe2x80x2 are independently as defined above;
R2 and R3 are independently chosen from C1-10alkyl, C1-10alkoxy, C2-10alkenyl, C2-10alkenyloxy, C2-10alkynyl or C2-10alkynyloxy; phenyl; naphthyl; a five-membered heteroaromatic ring containing 1, 2, 3 or 4 heteroatoms independently chosen from O, N and S, at most one of the heteroatoms being O or S; a six-membered heteroaromatic ring containing 1, 2 or 3 nitrogen atoms; and a group (CH2)pQ1 wherein Q1 is phenyl, naphthyl, a five-membered heteroaromatic ring containing 1, 2, 3 or 4 heteroatoms independently chosen from O, N and S, at most one of the heteroatoms being O or S, and a six-membered heteroaromatic ring containing 1, 2 or 3 nitrogen atoms; and wherein each of R2 and R3 is independently optionally substituted by one to three groups independently chosen from:
(a) halogen, cyano and nitro,
(b) hydroxy,
(c) C1-3alkyl, C2-3alkenyl and C2-3alkynyl,
(d) C1-3alkoxy,
(e) NR6R7 wherein R6 and R7 are independently as defined above,
(f) CO2R8 wherein R8 is independently as defined above,
(g) CONR6R7 or OCONR6R7 wherein R6 and R7 are independently as defined above,
(h) SO2NR6R7 wherein R6 and R7 are independently as defined above,
(i) CH2NR6R7 wherein R6 and R7 are independently as defined above,
(j) N(R8)COR8xe2x80x2 wherein R8 and R8xe2x80x2 are independently as defined above,
(k) NR8SO2R8xe2x80x2 where R8 and R8xe2x80x2 are independently as defined above;
alternatively R3 may be hydrogen;
R4 and R5 are independently chosen from hydrogen, C1-6alkyl optionally substituted by halogen, hydroxy, thiol, amino, C1-4alkoxy, C1-4 alkylthio, carboxy, C1-4 alkoxycarbonyl and (CH2)qQ2 wherein Q2 is a five-membered unsaturated heterocycle containing 1, 2, 3 or 4 heteratom optionally chosen from O, N, and S providing that not more than one heteroatom is O or S, a six-membered unsaturated heterocycle containing 1, 2 or 3 N atoms and phenyl and naphthyl, or a fused ring which is indolyl, each of the foregoing rings being optionally substituted with one to three groups independently chosen from hydroxy, C1-4alkyl, C1-4alkoxy, thiol, C1-4alkylthio, halogen, amino, carboxy, amido, CO2H and xe2x80x94NHC(NH2)2 and wherein each of the foregoing rings is optionally fused to a benzene ring; and
A is:
(1) hydrogen;
(2) C1-10alkyl, C2-10alkenyl or C2-10alkynyl optionally substituted with one to three substituents independently chosen from:
(i) hydroxy;
(ii) carboxy;
(iii) halogen;
(iv) C1-4alkoxy;
(v) C1-4alkoxycarbonyl;
(vi) xe2x80x94NR6R7 wherein R6 and R7 are independently chosen from hydrogen, C1-5alkyl and C1-5alkoxyC1-5alkyl;
(vii) xe2x80x94CONR6R7 or OCONR6R7 wherein R6 and R7 are independently as defined above;
(viii) xe2x80x94N(R8)QR9 wherein:
Q is C(O), C(S), SO2 or C(NH2);
R8 is hydrogen or C1-4alkyl; and
R9 is hydrogen, C1-4alkyl, C1-4alkoxy, amino, C1-4alkylamino di(C1-4alkyl)amino wherein each alkyl group is independently chosen;
(ix) C3-7cycloalkyl;
(x) phenyl or naphthyl; a five-membered heterocyclic ring containing 1, 2, 3 or 4 heteroatoms independently chosen from O, N and S, at most one of the heteroatoms being O or S; a six-membered heterocyclic ring containing 1, 2 or 3 nitrogen atoms; each of which is optionally substituted by one to three groups independently chosen from:
(a) halogen, cyano and nitro,
(b) hydroxy,
(c) C1-4alkyl, C2-4alkenyl and C2-4alkynyl,
(d) C1-4alkoxy,
(e) NR6R7 wherein R6 and R7 are independently as defined above,
(f) CO2R8 wherein R8 is independently as defined above,
(g) CONR6R7 or OCONR6R7 wherein R6 and R7 are independently as defined above,
(h) SO2NR6R7 wherein R6 and R7 are independently as defined above,
(i) CH2NR6R7 wherein R6 and R7 are independently as defined above,
(j) N(R8)COR8xe2x80x2 wherein R8 is independently as defined above and R8xe2x80x2 is independently as defined for R8, and
(k) NR8SO2R8xe2x80x2 wherein R8 and R8xe2x80x2 are independently as defined above; or
(3) a seven-membered heterocycle
having an otherwise unsubstituted carbon atom at the point of attachment to the rest of the compound of formula I,
having at a first atom alpha to the point of attachment a carbon atom which is unsubstituted or substituted by an oxygen or sulphur atom,
having at a first atom beta to the point of attachment, which atom is alpha to the foregoing first atom alpha, a carbon atom or a nitrogen atom,
having at a second atom alpha to the point of attachment a carbon atom, which is optionally substituted by oxygen, or a nitrogen atom,
having at a second atom beta to the point of attachment, which atom is alpha to the foregoing second atom alpha, a carbon atom or a nitrogen atom,
and having at the two remaining atoms carbon atoms;
a double bond may be present between the second atom alpha and the second atom beta;
the seven-membered heterocycle may be fused to one or two aromatic rings via any adjacent pair of atoms other than the point of attachment and the first atom alpha alone or in combination;
the aromatic ring may be benzene or a five-membered heterocycle containing 1, 2, 3 or 4 heteroatoms chosen from O, N and S providing that not more than one heteroatom is O or S or a six-membered heterocycle containing 1, 2 or 3 nitrogen atoms;
alternatively a pair of adjacent carbon atoms in the seven-membered heterocycle, other than the point of attachment and the first atom alpha alone or in combination, may form part of a fused cyclopropyl or cyclopentyl ring;
one to three substitutable atoms of the seven-membered heterocycle are optionally substituted by:
an aromatic ring as defined above optionally substituted by hydroxy, halogen, methoxy or alkyl having one to four carbon atoms;
an alkyl group having one to four carbon atoms optionally substituted by a halogen atom, hydroxy, an aromatic ring as defined above optionally substituted by hydroxy, halogen, methoxy or alkyl having one to four carbon atoms, cycloalkyl having three to seven carbon atoms, methoxy, bicycloalkyl having seven to twelve carbon atoms, heterocycle having five to seven atoms one of which is oxygen, nitrogen or sulphur which is optionally oxidized;
a heterocycle having five to seven atoms one of which is oxygen, nitrogen or sulphur which is optionally oxidized;
cycloalkyl having three to seven carbon atoms;
or bicycloalkyl having seven to twelve carbon atoms;
or the two groups A attached to the same nitrogen atom, together with that atom, form: a five-membered heterocyclic ring optionally containing 1, 2 or 3 further heteroatoms chosen from O, N and S, not more than one of the heteroatoms being O or S; or a six-membered heterocyclic ring optionally containing 1 or 2 further nitrogen atoms; each of which is optionally substituted by one to three groups independently chosen from:
(a) halogen, cyano and nitro,
(b) hydroxy,
(c) C1-4alkyl, C2-4alkenyl and C2-4alkynyl,
(d) C1-4alkoxy,
(e) NR6R7 wherein R6 and R7 are independently as defined above,
(f) CO2R8 wherein R8 is independently as defined above,
(g) CONR6R7 or OCONR6R7 wherein R6 and R7 are independently as defined above,
(h) SO2NR6R7 wherein R6 and R7 are independently as defined above,
(i) CH2NR6R7 wherein R6 and R7 are independently as defined above,
(j) N(R8)COR8xe2x80x2 wherein R8 is independently as defined above and R8xe2x80x2 is independently as defined for R8, and
(k) NR8SO2R8xe2x80x2 wherein R8 and R8xe2x80x2 are independently as defined above;
B is Cxe2x95x90O or CHOH in the R configuration;
X is oxygen or a bond;
n is zero or one, and
p is zero, one, two or three; and
q is zero, one, two or three;
with the proviso that no carbon atom is substituted by more than one hydroxy group.
In an embodiment the compounds of the present invention are of formula Ixe2x80x2: 
where R1, R2, R3, R4, R5, A and n are as defined above.
In one embodiment the compounds of the present invention are of formula Ixe2x80x3: 
where R1, R2, R3, R4, R5, A and n are as defined above.
In another embodiment there are provided compounds of formula Ixe2x80x2xe2x80x3: 
where R1, R2, R3, R4 and R5 are as defined above.
The following preferred definitions of substituents apply to each of the formulae I, Ixe2x80x2, Ixe2x80x3 and Ixe2x80x2xe2x80x3 which refer to those substituents.
Preferably R1 is
(1) C1-10alkyl, C2-10alkenyl or C2-10alkynyl optionally substituted with one to three substituents independently chosen from:
(i) hydroxy;
(ii) halogen;
(iii) amino;
(iv) C1-4alkoxy; and
(v) phenyl which is optionally substituted by one or two groups independently chosen from:
(a) halogen, cyano and nitro,
(b) hydroxy,
(c) C1-4alkyl, C2-4alkenyl and C2-4alkenyl,
(d) C1-4alkoxy and
(e) amino; or
(2) phenyl which is optionally substituted by one or two groups independently chosen from:
(a) halogen, cyano and nitro,
(b) hydroxy,
(c) C1-4alkyl, C2-4alkenyl and C2-4alkynyl,
(d) C1-4alkoxy and
(e) amino.
When R1 is a heterocyclic ring it may be saturated, partially saturated or unsaturated. Preferably the heterocyclic ring is a heteroaromatic ring.
More preferably R1 is C1-10alkyl optionally substituted with up to three substituents as defined above. Even more preferably R1 is C1-6alkyl optionally substituted by one to three substituents as defined above. Most preferably R1 is C1-6alkyl optionally substituted by halogen, phenyl, hydroxy or C1-4alkoxy. In particular R1 may be tertiary butyl or benzyl.
R2 and R3 may be independently chosen from phenyl; naphthyl; a five-membered heteroaromatic ring containing 1, 2, 3 or 4 heteroatoms independently chosen from O, N and S, at most one of the heteroatoms being O or S; a six-membered heteroaromatic ring containing 1, 2 or 3 nitrogen atoms; and a group (CH2)pQ1 wherein Q1 is phenyl; naphthyl; a five-membered heteroaromatic ring containing 1, 2, 3 or 4 heteroatoms independently chosen from O, N and S, at most one of the heteroatoms being O or S; and a six-membered heteroaromatic ring containing 1, 2 or 3 nitrogen atoms; and wherein each of R2 and R3 is independently optionally substituted by one to three groups independently chosen from:
(a) halogen, cyano and nitro,
(b) hydroxy,
(c) C1-3alkyl, C2-3alkenyl and C2-3alkynyl,
(d) C1-3alkoxy,
(e) NR6R7 wherein R6 and R7 are independently as defined above,
(f) CO2R8 wherein R8 is independently as defined above,
(g) CONR6R7 wherein R6 and R7 are independently as defined above,
(h) SO2NR6R7 wherein R6 and R7 are independently as defined above,
(i) CH2NR6R7 wherein R6 and R7 are independently as defined above,
(j) N(R8)COR8xe2x80x2 wherein R8 and R8xe2x80x2 are independently as defined above,
(k) NR8SO2R8xe2x80x2 where R8 and R8xe2x80x2 are independently as defined above;
More preferably R2 and R3 are (CH2)pQ1.
Preferably p is one.
Preferably Q1 is phenyl optionally substituted by one or two groups independently chosen from:
(a) halogen, cyano and nitro,
(b) hydroxy,
(c) C1-4alkyl, C2-4alkenyl and C2-4alkynyl,
(d) C1-4alkoxy and
(e) amino.
In one embodiment R2 and R3 are both benzyl.
More preferably Q1 is phenyl.
Preferably R4 and R5xe2x80x2 are independently chosen from optionally substituted C1-6alkyl and (CH2)qQ2. More preferably R4 and R5 are independently chosen from C1-6alkyl and (CH2)qQ2.
Preferably Q2 is optionally substituted phenyl. More preferably Q2 is phenyl.
In particular R4 and R5 are independently chosen from methyl, benzyl, phenyl, 2-methylpropyl, 1-hydroxyethyl, isopropyl and isobutyl.
A is preferably hydrogen or a group 
wherein R7 is phenyl, C1-6alkyl or C3-7cycloalkyl and Y is hydrogen or C1-6 alkyl. More preferably A is hydrogen or a group as defined above wherein R7 is a cyclohexyl group.
X is preferably oxygen.
n may be zero. n may be one.
p is preferably one.
q is preferably zero or one.
Thus a subclass of compounds of formula I and Ixe2x80x2 is provided wherein:
R1 is
(1) C1-10alkyl, C2-10alkenyl or C2-10alkynyl optionally substituted with one or more substituents independently chosen from:
(i) hydroxy;
(ii) halogen;
(iii) amino;
(iv) C1-4alkoxy; and
(v) phenyl which is optionally substituted by one or two groups independently chosen from:
(a) halogen, cyano and nitro,
(b) hydroxy,
(c) C1-4alkyl, C2-4alkenyl and C2-4alkynyl,
(d) C1-4alkoxy and
(e) amino; or
(2) phenyl which is optionally substituted by one or two groups independently chosen from:
(a) halogen, cyano and nitro,
(b) hydroxy,
(c) C1-4alkyl, C2-4alkenyl and C2-4alkynyl,
(d) C1-4 alkoxy and
(e) amino;
R2 and R3 are both (CH2)pQ1 wherein Q1 is phenyl optionally substituted by one or two groups independently chosen from:
(a) halogen,
(b) hydroxy,
(c) C1-4alkyl, C2-4alkenyl and C2-4alkynyl,
(d) C1-4alkoxy and
(e) amino;
R4 and R5 are independently chosen from C1-6alkyl optionally substituted by halogen, hydroxy, amino or C1-4alkoxy and (CH2)qQ2 wherein Q2 is phenyl optionally substituted by hydroxy, C1-4alkyl, C1-4alkoxy, thiol, C1-4alkylthio, halogen, amino, carboxy, amido, CO2H and xe2x80x94NHC(NH2)2;
A is hydrogen or 
wherein R7 is phenyl, C1-6alkyl or C3-7cycloalkyl;
Y is hydrogen or C1-6 alkyl;
n is zero or one;
p is one; and
q is zero or one.
For the avoidance of doubt each time the moieties A, R6, R7, R8, R8xe2x80x2 and R9 occur more than once in the definition of the compounds of formula (I) they are chosen independently.
As used herein, the expression xe2x80x9cC1-10alkylxe2x80x9d includes methyl and ethyl groups, and straight-chained and branched propyl, butyl, pentyl and hexyl groups. Particular alkyl groups are methyl, ethyl, n-propyl, isopropyl and t-butyl. Derived expressions such as xe2x80x9cC1-6alkylxe2x80x9d, xe2x80x9cC1-4alkylxe2x80x9d, xe2x80x9cC2-10alkenylxe2x80x9d, xe2x80x9cC2-4alkenylxe2x80x9d, xe2x80x9cC2-10 alkynylxe2x80x9d and xe2x80x9cC2-4alkynylxe2x80x9d are to be construed in an analogous manner.
The expression xe2x80x9cC3-7cycloalkylxe2x80x9d as used herein includes cyclic propyl, butyl, pentyl, hexyl and heptyl groups such as cyclopropyl and cyclohexyl.
The term xe2x80x9cheterocyclicxe2x80x9d includes rings which are saturated, partially saturated or unsaturated. Unsaturated heterocyclic rings are also known as heteroaromatic rings.
Suitable 5- and 6-membered heteroaromatic rings include pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, imidazolyl, oxadiazolyl and thiadiazolyl groups. A suitable 5-membered heteroaromatic ring containing four nitrogen atoms is tetrazolyl. Suitable 6-membered heteroaromatic rings containing three nitrogen atoms include 1,2,4-triazine and 1,3,5-triazine. Suitable saturated heterocyclic rings include piperazine, morpholine, piperidine, tetrahydrofuran and tetrahydrothiophene. Tetrahydrofuran is preferred.
The term xe2x80x9chalogenxe2x80x9d as used herein includes fluorine, chlorine, bromine and iodine, of which fluorine and chlorine are preferred.
As used herein the term xe2x80x9cC1-4alkoxyxe2x80x9d includes methoxy and ethoxy groups, and straight-chained, branched and cyclic propoxy and butoxy groups, including cyclopropylmethoxy.
Specific Examples according to the present invention include:
{1S-benzyl-4R-[1-(1S-carbamoyl-2-(R)-hydroxypropylcarbamoyl)-(S)-ethylcarbamoyl]-2R-hydroxy-5-phenylpentyl}-carbamic acid, tert-butyl ester;
{1S-benzyl-4R-[1-(1S-carbamoyl-2-(S)-methylbutylcarbamoyl)-1S-2-methylpropylcarbamoyl]-2R-hydroxy-5-penylpentyl}carbamic acid tert-butyl ester;
{1S-benzyl-4-[1-(5-cyclohexyl-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3(R,S)-yclarbamoyl)-(S)-ethylcarbamoyl]-2R-hydroxybutyl}-carbamic acid, tert-butyl ester;
{1S-benzyl-4R-[1-(5-cyclohexyl-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3(R,S)-yclarbamoyl)-(S)-ethylcarbamoyl]-2R-hydroxy-5-phenylpentyl}-carbamic acid, tert-butyl ester;
{1S-benzyl-4R-[1-(1S-carbamoyl-2-phenylethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2R-hydroxy-5-phenylpentyl}-carbamic acid, benzyl ester;
{1S-benzyl-4R-[1-(1S-carbamoyl-2-phenylethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2-oxo-5-phenylpentyl}-carbamic acid tert-butyl ester;
{1S-benzyl-4R-[1-(1S-carbamoyl-2-phenylethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2R-hydroxy-5-phenylpentyl}carbamic acid tert-butyl ester;
{1S-benzyl-4R-[1-(1S-carbamoyl-2-phenylethylcarbamoyl)-S-ethylcarbamoyl]-2R-hydroxy-5-phenylpentyl}carbamic acid tert-butyl ester;
(1S-benzyl-4R-{1S-[(carbamoylphenylmethyl)carbamoyl]-S-ethylcarbamoyl}-2R-hydroxy-5-phenylpentyl)carbamic acid tert-butyl ester; and the pharmaceutically acceptable salts thereof.
Examples of pharmaceutically acceptable salts are hydrochlorides, sulfates, citrates, tartrates, acetates, methanesulfonates, phosphates, oxalates and benzoates.
The compounds of the present invention have an activity as inhibitors of xcex3 secretase. In a preferred embodiment the compounds of the invention inhibit proteolysis of PS-1.
The invention also provides pharmaceutical compositions comprising one or more compounds of this invention and a pharmaceutically acceptable carrier. Preferably these compositions are in unit dosage forms such as tablets, pills, capsules, powders, granules, sterile parenteral solutions or suspensions, metered aerosol or liquid sprays, drops, ampoules, transdermal patches, auto-injector devices or suppositories; for oral, parenteral, intranasal, sublingual or rectal administration, or for administration by inhalation or insufflation. For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical carrier, e.g. conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums or surfactants such as sorbitan monooleate, polyethylene glycel, and other pharmaceutical diluents, e.g. water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. Typical unit dosage forms contain from 1 to 100 mg, for example 1, 2, 5, 10, 25, 50 or 100 mg, of the active ingredient. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
The present invention also provides a compound of the invention or a pharmaceutically acceptable salt thereof for use in a method of treatment of the human body. Preferably the treatment is for a condition associated with the deposition of xcex2-amyloid. Preferably the condition is a neurological disease having associated xcex2-amyloid deposition such as Alzheimer""s disease.
The present invention further provides the use of a compound of the present invention or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating or preventing Alzheimer""s disease.
Also disclosed is a method of treatment of a subject suffering from or prone to Alzheimer""s disease which comprises administering to that subject an effective amount of a compound according to the present invention or a pharmaceutically acceptable salt thereof.
The liquid forms in which the novel compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions, suitably flavoured syrups, aqueous or oil suspensions, and flavoured emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinyl-pyrrolidone or gelatin.
For treating or preventing Alzheimer""s Disease, a suitable dosage level is about 0.01 to 250 mg/kg per day, preferably about 0.01 to 100 mg/kg per day, and especially about 0.01 to 5 mg/kg of body weight per day. The compounds may be administered on a regimen of 1 to 4 times per day. In some cases, however, dosage outside these limits may be used.
There is also provided a process for producing a compound of formula I or a pharmaceutically acceptable salt thereof which comprises reacting a compound of formula II with a compound of formula III: 
wherein R1, R2, R3, R4, R5, A, X and n are as defined above and P is hydrogen or a protecting group such as a trialkylsilane group, for example t-butyl dimethylsilyl, followed, if necessary, by deprotection of the resulting compound to produce a compound of formula I. The reaction is generally carried out in the presence of coupling agents such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole in a solvent such as DMF, generally at about room temperature for six to twelve hours. Any necessary deprotection is achieved by conventional means.
The compound of formula II is produced by reacting a compound of formula IV: 
wherein R1, R2, R3 and X are as defined above in a solvent such as dioxane, with a base such as lithium hydroxide in a polar solvent such as water generally at room temperature for above five hours. If desired the resulting compound of formula II in which P is hydrogen is protected by conventional means.
The compound of formula IV is produced by reacting a compound of formula V with a compound of formula VI or a compound of formula VII: 
wherein R1, R2 and X are as defined above, R3xe2x80x2 is the acyl derivative of a group R3 as defined above and R3xe2x80x3 is a group R3 bound to an oxo group at the portion of R3 which connects to the compound of formula V. The reaction is generally carried out in the presence of a base such as lithium diisopropylamide in a solvent such as THF generally cooled to xe2x88x9278xc2x0 C. for about thirty minutes. The reaction mixture is subsequently dehydrated without purification and then hydrogenated with, for example, hydrogen over 5% Pd/C catalyst at about 50 psi for about 2 h.
The compound of formula V in which R1 is tertiary butyl and R2 is benzyl can be prepared as described by J. Litera et al., Collect. Czech. Chem. Commun. 1998, 63, 231ff. Compounds of formula V in which R1 is other than tertiary butyl and R2 is other than benzyl can be made by analogous methods.
Compounds of formulae III and VI are commercially available or known in the prior art or can be made from commercially available or known compounds by standard methods.
Alternatively the compounds of the present invention can be made by the following typical experimental procedure or by methods analogous thereto: 
in which R1, R2, R3, R4, R5 and n are as defined above and A is hydrogen.
A. 50 mg (0.030 mmol) of FMOC-Sieber amide resin is placed in a Quest 210 solid phase reactor and treated with Piperidine/DMF (0.5 mL; 1:1 mixture) with mixing for 30 minutes. The reactor is drained and washed with DMA (10xc3x971 mL). 1 mL of a 0.1 M solution of FMOC-amino acid 1 in DMA is added followed by 0.2 mL of a DMA mixed solution of HOBT and Hunig""s base (0.5 M in both) and 0.5 mL of a 0.2 M solution of PyBOP in DMA. The reactor is mixed for 60 minutes, drained, and washed with DMA (10xc3x971 mL). The reactor is treated with Piperidine/DMF (0.5 mL; 1:1 mixture) and mixed for 30 minutes. The reactor is drained and washed with DMA (10xc3x971 ML). 1 mL. of a 0.1 M solution of FMOC-amino acid 2 in DMA is added followed by 0.2 mL of a DMA mixed solution of HOBT and Hunig""s base (0.5 M in both) and 0.5 mL of a 0.2 M solution of PyBOP in DMA. The reactor is mixed for 60 minutes, drained, and washed with DMA (10xc3x971 mL). The reactor is treated with Piperidine/DMF (0.5 mL; 1:1 mixture) and mixed for 30 minutes. The reactor is drained and washed with DMA (10xc3x971 mL).
B. 21 mg (0.04 mmol) of the TBS-protected isostere and 5 mg HOBT in 0.5 mL NMP is added to the reactor, followed by Hunig""s base (50 xcexcL) and 0.5 M PyBOP in NMP (0.5 mL). The reactor is mixed for 16 h, filtered, and washed with DMA (5xc3x971 mL), MeOH (2xc3x971 mL) and DCE (10xc3x971 mL).
C. The reactor is treated with 0.5 mL of a 1% solution of TFA in DCM and stood for 10 minutes. The reactor is drained into a test tube containing 2 M NH3 in MeOH (1 mL) and the cleavage repeated a further 5 times (identical conditions). The product is concentrated (SpeedVac), dissolved in 1 mL DCM and washed with water (1 mL) using a phase separation CombiTube. The DCM layer is concentrated (SpeedVac) to give essentially pure TBS-protected product.
D. The product is dissolved in 1 M TBAF in THF (0.5 mL), stood for 7 h, water (2 mL) is added and the product is extracted out with DCM (3xc3x971 mL). The product is purified by flash chromatography (5%MeOH/DCM).
All products are analyzed by analytical LC-MS utilizing diode array detection (210-250 nm) and APcI detection (150-850 amu) using a full 5%xe2x86x9295% MeCN gradient with 0.1% aqueous TFA. A strong M+Na+ peak is oberved in the mass spectrum.
It will be understood that any compound of formula I initially obtained from the above process may, where appropriate, subsequently be elaborated into a further compound of formula I by techniques known from the art.
It will also be appreciated that where more than one isomer can be obtained from a reaction then the resulting mixture of isomers can be separated by conventional means.
Where the above-described process for the preparation of the compounds according to the invention gives rise to mixtures of stereoisomers, these isomers may be separated by conventional techniques such as preparative chromatography. The novel compounds may be prepared in racemic form, or individual enantiomers may be prepared either by enantiospecific synthesis or by resolution.
During any of the above synthetic sequences it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups, such as those described in Protective Groups in Organic Chemistry, ed. J. F. W. McOmie, Plenum Press, 1973; and T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, John Wiley and Sons, 1991. The protecting groups may be removed at a convenient subsequent stage using methods known from the art.
A typical assay which can be used to determine the level of activity of compounds of the present invention is as follows:
(1) Mouse neuroblastoma neuro 2a cells expressing human app695 are cultured at 50-70% confluency in the presence of sterile 10 mM sodium butyrate.
(2) Cells are placed in 96-well plates at 30,000/well/100 xcexcL in minimal essential medium (MEM) (phenol red-free) +10% foetal bovine serum (FBS), 50 mM HEPES buffer (pH7.3), 1% glutamine, 0.2 mg/mL G418 antibiotic, 10 mM sodium butyrate.
(3) Make dilutions of the compound plate. Dilute stock solution to 5.5% DMSO/110 xcexcM compound. Mfix compounds vigorously and store at 4xc2x0 C. until use.
(4) Add 10 xcexcL compound/well. Mix plate briefly, and leave for 18 h in 37xc2x0 C. incubator.
(5) Remove 90 xcexcL of culture supernatant and dilute 1:1 with ice-cold 25 mM HEPES (pH.3), 0.1% BSA, 1.0 mM EDTA (+broad spectrum protease inhibitor cocktail; pre-aliquotted into a 96-well plate). Mix and keep on ice or freeze at xe2x88x9280xc2x0 C.
(6) Add back 100 xcexcL of warm MEM +10% FBS, 50 mM HEPES (pH7.3), 1% glutamine, 0.2 mg/mL G418, 10 mM sodium butyrate to each well, and return plate to 37xc2x0 C. incubator.
(7) Prepare reagents necessary to determine amyloid peptide levels, for example by ELISA assay
(8) To determine if compounds are cytotoxic cell viability following compound administration is assessed by the use of redox dye reduction. A typical example is a combination of redox dye MTS (Promega) and the electron coupling reagent PES. This mixture is made up according to the manufacturer""s instructions and left at room temperature.
(9) Quantitate amyloid beta 40 and 42 peptides using an appropriate volume of diluted culture medium by standard ELISA techniques.
(10) Add 15 xcexcL/well MTS/PES solution to the cells; mix and leave at 37xc2x0 C.
(11) Read plate when the absorbance values are approximately 1.0 (mix briefly before reading to disperse the reduced formazan product).
The Examples of the present invention all had an ED50 of less than 500 nM, preferably less than 200 nM and most preferably less than 100 nM in the above assay.